Von Hippel-Lindau disease is a hereditary cancer syndrome characterized by an increased risk of central nervous system tumors (hemangioblastomas), kidney cancer, and adrenal gland tumors (pheochromocytomas). This disorder arises in individuals who harbor a defective copy of the VHL tumor suppressor gene. VHL inactivation is also common in non-hereditary kidney cancers and hemangioblastomas. The best understood function of the VHL gene product (pVHL) relates to 'HIF', which is a master transcriptional regulator of genes involved in adaptation to hypoxia. HIF consists of two proteins, an 'alpha'subunit and a 'beta'subunit. pVHL targets HIFalpha for destruction when oxygen is present. The physical interaction of pVHL with HIFalpha requires that HIFalpha be hydroxylated on one of two prolyl residues by EglN1, which is an oxygen-dependent enzyme. pVHL-defective cells fail to destroy HIFalpha and consequently overexpress various HIF-responsive genes. There are 3 HIFalpha family members although HIF2alpha appears to be most relevant with respect to kidney cancer. Specific aim 1 will ask whether HIFalpha (especially HIF2alpha) stabilization in the mouse is sufficient to cause the pathological changes observed when pVHL is defective. Some VHL mutations cause very different risks of kidney cancer despite sharing HIFalpha defects. Specific aim 2 will attempt to understand the basis for this differential risk using cell biological, genomic, and biochemical approaches. Some VHL mutations preserve the ability to regulate HIF and yet cause pheochromocytoma. We recently found that all of the genes linked to familial pheochromocytoma (VHL, NF1, c-Ret, SDHB, SDHC, SDHD) regulate apoptosis of sympathoadrehal precursor cells following NGF withdrawal and that pheochromocytoma-associated alleles allow cells to escape cell death in this setting. Apoptosis after NGF withdrawal requires EglN3, a paralog of the HIF prolyl hydroxylase EglN1. Specific aim 3 will attempt to elucidate how EglN3 induces apoptosis. Many human cancers are driven by excessive Cyclin D1 activity. In the fly, elimination of the sole EglN family member impairs Cyclin D-dependent proliferation. In aim 4 we will pursue our preliminary data that loss of EglN2, the third member of the mammalian EglN family, downregulates Cyclin D1 in vitro and in vivo. This work is relevant to many human cancers and has already motivated successful clinical trials of agents that inhibit HIF-responsive growth factors (such as VEGF) in kidney cancer. Work in this area has also provided new insights into how human cells 'sense'and respond to inadequate oxygen availability. This, in turn, is leading to new drugs that might be useful in diseases such as heart attack and stroke that are characterized by impaired oxygen delivery to tissues.